The present invention relates to methods and apparatus for delivery of pharmaceuticals to target tissues in situ, in vivo, ex vivo, or in vitro.
Advances in recombinant-DNA technology have made introduction of therapeutic genes into somatic cells possible (Anderson W F, Human gene Therapy. Science 256:808-813, 1992; Miller A D, Human gene therapy comes of age. Nature 357:455-457, 1992). In recent years several clinical trials involving human gene therapy have been accepted by regulatory agencies. The first of the approved clinical trials which have initiated human gene therapy aim at treating both inherited diseases (such as severe combined immunodeficiency caused by lack of adenosine deaminase in peripherial T-lymphoctes, cystic fibrosis, and familial hypercholesterolemia) as well as noninherited disease such as cancer (Wolfe J H, Recent progress in gene therapy for inherited disease. Curr. Opinion in Pediatr. 6:213-219, 1994; Sanda M G et al., Gene therapy for urologic cancer. J. Urology 44:617-624, 1994; O""Malley B W et al., Somatic gene therapy in otolaryngology head and neck surgery. Arch. Otolaryngol. Head Neck Surgery 119:1191-1197, 1993; Engelhardt J F et al., Direct gene transfer of human CFTR into human bronchial epithelia of xenografts with Ei-deleted adenoviruses. Nature Genetics 4:27-34, 1993; Lemarchand P et al., Adenovirus-mediated transfer of recombinant human alphal-antitrypsin cDNA to human endothelial cells. PNAS (USA) 89:6482-6486, 1992; Jaffe H A et al., Adenovirus-mediated in vivo gene transfer and expression in normal rat liver. Nature Genetics 1:372-378, 1992).
The development of suitable, safe and effective gene transfer systems is a major goal of research in gene therapy. Thus far, viruses have been extensively used as vectors for gene therapy. For example, retroviruses have been widely used, but a major disadvantage is that they can only be used as vectors which target actively dividing cells. In addition, retroviruses do not accomodate large DNA inserts readily. Adeno-associated viruses are also limited in the ability to accomodate large inserts, yet replication defective adenovirus has been successfully used for transfer of a variety of genes into cells in culture and in vivo. Adenoviruses can accomodate larger inserts than retroviruses, but extrachromasomal expression usually only lasts for a few weeks. Herpes viruses have been exploited for specific gene transfer trials into the central nervous system. Herpes viruses can carry large foreign DNA inserts, and may remain latent for long periods of time. In spite of the availability of replication defective viruses, concerns about the safety and efficiency of such viral vectors has generated interest in the development of nonviral gene transfer systems such as liposome-DNA complexes and receptor mediated endocytosis (Felgner P L et al., PNAS(USA) 84: 7413-7417, 1987; Hyde, Nature 362: 250-255, 1993; Nu GY. J. Biol. Chem. 266: 14338, 1991).
A major hurdle for effective gene therapy is the development of methods for targeting the gene transfer to appropriate target cells and tissues. Ex vivo gene transfer into explanted cultured cells and implantation of the treated cells has been used for the treatment of hematopoietic tissues (U.S. Pat. No. 5,399,346, issued Mar. 21, 1995, Anderson et al., hereby incorporated by reference). Direct injection into tissues, intravenous or intra-arterial administration, inhalation, or topical application have also been used. Major drawbacks to all of these methods are that the transduction is not highly selective, significant amounts of the therapeutic gene containing vector may be needed, and efficency of the gene transfer is severely limited by the constraints of vector concentration, time of exposure to the target, and effectiveness of the gene transfer vector.
One area of active research has been gene therapy into mammalian kidneys, but the results have been disappointing because of problems with efficiency of gene transfer (Woolf A S et al., Gene transfer into the mammalian kidney: First steps towards renal gene therapy. Kidney Int. 43: Suppl. 39: S116-S119, 1993). Moullier et al. (Adenoviral-mediated gene transfer to renal tubular cells in vivo. Kidney Int. 45: 1220-1225, 1994), showed some adenovirus-mediated transfer of lacZ gene into rat tubular but not glomerular cells following a combination of infusion of the virus into the renal altery and retrograde infusion into the vector. Simple infusion of soluble virus does not appear to be an efficient transfer system. Better results were obtained by Tomita et al., (Direct in vivo gene introduction into rat kidney. Biochem. Biophys. Res. Commun. 186: 129-134, 1992), who infused a complex of Sendai virus and liposomes into the rat renal artery in vivo. This resulted in expression of the marker gene in about 15% of the glomerular cells.
It would be useful to the medical arts, to be able to have apparatus and methods for the efficient administration of gene therapy to target cells and tissues which overcomes the limitations inherent to each gene transfer vector.
In accordance with an aspect of the present invention, there is provided methods for the administration of pharmaceuticals to targets for functional use. (The term xe2x80x9cpharmaceutical,xe2x80x9d as used herein, includes chemical drugs, protein drugs, nucleic acid drugs, combination chemical/protein/nucelic acid drugs, and gene therapy vectors. The term xe2x80x9cfunctional use,xe2x80x9d as used herein, includes therapeutic treatment, prophylaxis, and/or production of recombinant proteins in vivo. The term xe2x80x9cfunctional usexe2x80x9d also includes the disruption of endogenous gene expression including the use of antisense, triplex forming, catalytic and otherwise disruptive pharmaceuticals. The term xe2x80x9cfunctional usexe2x80x9d also includes the expression of recombinant proteins in target tissues, whether of endogenous or exogenous origin. The term xe2x80x9ctarget,xe2x80x9d as used herein, includes cells, tissues and/or organs. The term xe2x80x9cgene therapy vectorxe2x80x9d is meant to include nucleic acid constructs which are single, double or triplex stranded, linear or circular, that are expressible or non-expressible constructs which can either encode for and express a functional protein, or fragment thereof, or interfere with the normal expression of a target gene, gene transfer and/or expression vectors.)
The administration of pharmaceuticals may take place where the target is in situ in a living subject. The administration may also take place wherein the target is first removed from a subject, manipulated ex vivo, and returned to the original or alternatively a second recipeint subject. In a preferred embodiment, the target is situated such that the circulation of the blood supply into and out of the target is relatively isolated. In a most preferred embodiment, the blood circulation into and out of the target is mostly via a single, or readily identified entering arteries and exiting veins. There are of course certain amounts of limited leakage due to small blood and lymphatic vessels.
The methods of the instant invention allow for a prolonged period of administration of pharmaceuticals to a target by way of recirculating a pharmaceutical containing solution through the target such that a perfusion effect occurs. The methods of the instant invention allow for prolonged administration because of the unique use of the perfusion method and the oxygenation of the pharmaceutical containing solution. In one embodiment, the perfusion apparatus and target forms a closed system whereby the pharmaceuticals are administered at a starting concentration and not adjusted during the time course of treatment. In another embodiment, the pharmaceutical concentration is periodically adjusted so as to maintain or otherwise alter the concentration of pharmaceutical in the solution, or additional pharmaceuticals are added. In a preferred embodiment, the solution does not require replenishment during the course of treatment. In another embodiment, the solution volume can be replenished as leakage or other forms of loss occurs during the course of treatment. (The term xe2x80x9csolution,xe2x80x9d as used herein refers to the medium in which the pharmaceutical is suspended, dissolved or otherwise maintained for delivery to the target, aka. the perfusate, and includes blood, serum, plasma, saline, and/or buffered solutions.) In a preferred embodiment, 350 ml of perfusate contains red blood cells (around 17% of hemocrit value), and can include about 25,000 IU heparin, about 20,000 IU penicillin and about 20,000 xcexcg streptomycin in Krebs-Ringer solution in addition to the pharmaceutical.
The instant invention also provides for a perfusion apparatus comprising functionally connected by a perfusate transfer system, (a) a reservoir for perfusate, (b) means for propelling the perfusate through the apparatus, (c) means for oxygenation of the perfusate, (d) means for connecting the apparatus to and from the target.
The reservoir for the perfusate can be any container which can be sterilized and used to collect perfusate from the target. The reservoir is connected to the means for transporting the perfusate through the system by means of tubing. While perfusion may occur at room temperature of 20xc2x0 C., in a preferred embodiment, the perfusion occurs at 37xc2x0 C. Thus, in practice, the perfusate reservoir can be maintained at any desired temperature via, for example, a water bath.
In an embodiment where the means for propelling the perfusate is a peristaltic pump, the tubing is preferably silicone or other such suitable pliable tubing. Where the means for propelling the perfusate is a peristaltic pump, no contact is made between the perfusate and any part of the pump directly. In the case where a pump with, for example an impeller blade is used, then the perfusate comes into direct contact with a part of the pump. In the usual configuration using a peristaltic pump, the tubing from the reservoir passes through the pump and connects with the means for oxygenating the perfusate.
The means for oxygenating the perfusate can be any form of artificial lung, or aeration device such that the perfusate is oxygenated without over agitation and susequent frothing. In one embodiment the means for oxygenating the perfusate is a membrane lung which consists of a length of semi-permeable tubing packed into a gas chamber into which is circulated oxygen rich gas for oxygenating the perfusate as it passes through the length of tubing. In a preferred embodiment, the membrane lung contains about 8 m of silicon tubing of approximately 1.47 mm inside diameter, and the gas circulated in the chamber is carbogen gas (comprised of 95% oxygen, 5% carbon dioxide).
In general, the target is cannulated and connected to tubing connecting from the means for oxygenating the perfusate, and leading to the perfusate reservoir. In one configuration, the perfusate is pumped from a reservoir, through a means for oxygenating the perfusate, into the target, through the target, and back into the reservoir. The location of the pumping means in relation to the other components can be varied. The number of each component can also be varied.
Thus the instant invention provides for a method of administering a pharmaceutical to a target whereby the target is mostly isolated and continuosly perfused with a perfusate containing the pharmaceutical, and said perfusate is recirculated and oxygenated. The instant invention provides for an apparatus for the administration of pharmaceuticals to a target comprising a perfusate reservoir, means for pumping the perfusate, means for oxygenating the perfusate, and means for connecting the components to one another, and with the target. In a preferred embodiment the recirculating perfusion apparatus comprises a perfusate reservoir receiving efflux perfusate from the target, connected with silicone tubing passing via a peristaltic pump to a membrane lung, said membrane lung comprising about 8 m of approximately 1.47 mm inner diameter silicone tubing immersed in a circulating gas chamber filled with carboxygen gas, connected by tubing and a catheter to a target.